1. Field of the Invention
This present invention relates to the field of cell biology of undifferentiated cells. More specifically, it relates to the propagation of undifferentiated cells, culture conditions and materials that facilitate propagation and use of undifferentiated cells.
2. Description of the Prior Arts
Undifferentiated cells, such as tissue progenitor cells, stem cells and the like, have great commercial potential in regenerative medicine or therapeutic tissue engineering. For the application of undifferentiated cells in regenerative medicine or therapeutic tissue engineering, a convenient method for culturing undifferentiated cells in an undifferentiated state in vitro is required.
Stem cells represent a generic group of undifferentiated cells and preserve the ability to renew themselves through cell division and can differentiate into different kinds of differentiated cells, and are found in all multiple cellular organisms. In mammals, three main of stem cells are embryonic stem cells that are found in blastocytes, extraembryonic stem cells are found in extraembryonic tissues, and postnatal stem cells that are found in postnatal tissues. The postnatal stem cells act as a repair system for replenishing specialized cells. As known in the field of the art, stem cells can propagate in culture in an undifferentiated state in the presence of feeder cells.
However, the potential risk of using feeder cells in the culture of the undifferentiated cells such as stem cells for regenerative medicine or therapeutic tissue engineering is that infectious agents such as viruses may infect the recipient. Therefore, there is a need for alternative method for culturing undifferentiated cells in vitro in an undifferentiated state in the absence of feeder cells.
Some of extracellular matrix components (ECM components) are used to replace the feeder cells for culturing undifferentiated cells to maintain them in an undifferentiated state. A few methods for culturing undifferentiated cells with ECM components such as laminin and collagen have been developed.
WO 98/50576 discloses a method of culturing neuroepithelial stem cells and oligodendrocyte-astrocyte precursor cells. It is observed that differentiation of the neuroepithelial stem cells into oligodendrocytes, astrocytes and neurons can be induced by replating the cells on laminin, withdrawing mitogens or adding dorsalizing agents to the growth medium.
WO 2008/007082 A2 discloses a method to maintain primate embryonic stem cells in cell culture conditions that are cell feeder free and serum free with a cell culture vessel coated with proteinaceous based cell culture support, wherein the proteinaceous based cell culture support is a collagen-based cell culture support.
Based on the foregoing, some ECM components are not only incapable of maintaining the differentiation potential of the cells, but induce the differentiation of the undifferentiated cells. It is suggested that the inconsistent effects of the different ECM components on the undifferentiated cells in culture result from the diverse properties of the different ECM components.
Hyaluronan (HA), one of the chief components of the extracellular matrix, is a non-sulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. HA is applied to postnatal stem cells and is reported to influence the cells on the migration, proliferation (S. K. Nilsson et al., (2003), Blood, 101: 856-862; D. Peck and C. M. Isacke, (1996), Curr Biol, 6: 884-890), and cell behavior (C. B. Knudson, (2003), Birth Defects C Embryo Today, 69:174-196) as well as the developmental capacity of bovine embryos in vitro (M. Stojkovic et al., (2002), Reproduction, 124: 141-153). The enhancement of osteogenic potential of rat osteoblasts by an initial administration of HA during first plating was also suggested (L. Huang et al., (2003), J Biomed Mater Res A, 66: 880-884). However, none of the above documents discloses that HA is able to maintain undifferentiated cells in culture, such as stem cells in an undifferentiated state.
In U.S. Pat. Application No. 20020042132, Gardner, David K. et al disclose a mammalian culture medium supplement comprising recombinant human albumin and fermented hyaluronan (HA) and a medium containing the supplement capable of increasing the viability of gametes or embryonic cells. Some other findings suggested that HA can stimulate the proliferation of primary porcine bone marrow stromal cells during early passage (X. Zou et al., (2004), Biomaterials, 25: 5375-5385). It is demonstrated that HA suspended in a medium stimulates rather than maintain proliferation capacity of cells.
Therefore the results from both the intrinsic difference between the proteinaceous ECM components and non-proteinaceous ECM components and the different means for introducing the ECM components to the undifferentiated cells are inconsistent.
New technology to manipulate the differentiation of undifferentiated cells, especially pluripotent stem cells would be a substantial achievement towards realizing the fall commercial potential of stem cell therapy, and will also be a very valuable means for medicine.